Regulation of adenovirus gene expression by the virus-coded 72K early protein is being studied in isolated nuclei. Manipulation of temperature and use of inhibitors of macromolecular synthesis prior to nuclear isolation are used to probe the specificity and mechanism of action of the 72K protein. Conditions that allow de novo initiation of RNA synthesis in vitro will be used to test the effect of the 72K protein on virus transcription, using both cell extracts and purified 72K protein. The action of TPA on adenovirus early transcription will also be investigated in the isolated nuclear system. Revertants of ts125 will be screened for ability to regulate early virus transcription, and will also be tested for ability to transform rat embryo fibroblasts. The relationship of the 72K protein to cell transformation will be further tested by measurement of transcription rate in vivo in rat embryo cells infected with various mutant alleles of the 72K protein gene. Investigation of selective procedures for isolating possible Ad5 regulatory mutants is continuing.